Hepatitis C virus (HCV) is the most prevalent chronic blood-borne infection in the U.S and a global health problem. Approximately 4 million individuals in the U.S. and 170 million individuals worldwide are chronically infected. As much as 40 percent of chronic liver disease is HCV related and this results in up to 10,000 deaths each year. Despite improvements, treatment of remains suboptimal, and new therapeutic options are critically needed. Apath, LLC has developed cell-based assays for testing and screening of compounds with antiviral activity against hepatitis C virus (HCV). These assays are based on Huh7 cell lines that contain a constructively replicating subgenomic HCV replicon. The cells have been analyzed extensively and have been demonstrated to contain an HCV replicon that exhibits autonomous HCV RNA replication. We have promoted these cell lines as drug-discovery tools by formatting assays for compound screening that are amenable to high throughput screening. In this Phase II proposal we describe our plans to use these cell-based assays to identify compounds with anti-HCV activity. Toward this goal we will plan to utilize a unique source of natural compounds and to screen over 40,000 wells containing up to 200,000 discrete compounds to identify "hits" that inhibit replication of the HCV replicon. We will confirm antiviral activity of all "hits" in secondary HCV replicon quantitation and cytotoxicity assays. We will identify compounds with the most promising chemical and biological profiles to pursue as potential anti-HCV drugs. We also plan to identify targets of leads through biochemical and genetic approaches.